Biosensors

Characterization of M11-like and OC125-like Monoclonal Antibody Binding to CA125 Tandem Repeats

The CA125 epitope within the MUC16 tandem repeat region is detected via the CA125 II test for ovarian cancer surveillance. This test utilizes the M11 and OC125 antibodies. A revised model of MUC16 with 19 tandem repeats has recently been identified, including splice variants that exclude entire repeats. Additionally, OC125 has exhibited gaps in coverage of the tandem repeat region. To identify antibodies that bind more repeats and are suitable for spliceoform detection, more antibodies must be characterized using the revised model. This study characterized the binding of two M11-like and two OC125-like antibodies against the updated tandem repeat numbering system. 16 individual tandem repeats were expressed and purified. Binding interactions between each of the antibodies and recombinant repeats were examined by indirect enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). The M11-like antibodies displayed different binding patterns when compared to each other, while the two OC125-like antibodies exhibited similar binding patterns. M11-like clone M77161 bound to all 16 repeats tested, indicating that it may be suitable for accurate detection of CA125. These findings demonstrate how different antibodies vary in their binding to CA125, contributing to ongoing development of improved clinical and research tools for ovarian cancer.

An Extracellular Vesicle (EV) Paper Strip for Rapid and Convenient Estimation of EV Concentration

Extracellular vesicles (EVs) have emerged as promising biomarkers and therapeutic agents, yet their quantification remains technically challenging due to the limitations of conventional methods. Here, a low-cost, fluorescence-based, paper-strip immunoassay is presented for rapid and semi-quantitative estimation of EV concentration, inspired by pH strips. The assay utilizes nitrocellulose membranes functionalized with capture antibodies (anti-CD63, CD9, CD81) and fluorescent dye (ExoBrite™) for EV detection. Systematic optimization of assay parameters—including dye application sequence, incubation time, antibody configuration, and dye concentration—revealed that labeling EVs with dye and incubating on the nitrocellulose paper strips for 20 min yielded the strongest and most reproducible signal. A 200× dilution of ExoBrite™ dye was determined to provide the best balance between sensitivity and specificity. A standard curve generated through twofold serial dilution of EVs from ovarian cancer cell culture medium confirmed a positive, concentration-dependent fluorescence response, establishing a usable dynamic range. Compared to existing technologies, this platform enables fast, simple-to-implement EV quantification using minimal sample volume and equipment. The simplicity and scalability of the method offer strong potential for use in clinical diagnostics and EV research applications.

Modern Emerging Biosensing Methodologies for the Early Diagnosis and Screening of Ovarian Cancer

Ovarian cancer (OC) is one of the leading causes of gynecological cancer-related death worldwide. Late diagnosis at advanced stages of OC is the reason for a higher mortality rate. Earlier diagnosis and proper treatment are important for improving the prognosis of OC patients. Biosensors offer accurate, low-cost, rapid, and user-friendly devices that can be employed for the detection of OC-specific biomarkers in the early stage. Therefore, it is important to consider the potential biomarkers in the biological fluids to confirm the OC prognosis. Out of many biomarkers, the most commonly tested clinically is cancer antigen 125 (CA-125). However, CA-125 is considered to be a poor biomarker for OC diagnosis. Several biosensing methods were developed for the sensitive and quantitative detection of each biomarker. In abnormal expression in OC patients, nucleic acids, enzymes, cells, and exosomes are used as target biomarkers for the construction of biosensors. This review focuses on the development for the detection of various biomarkers using multiple biosensing methods. Here, we describe the origin and the significance of OC-associated biomarkers, the working principle of biosensors, and the classification of biosensors based on their recognition elements and signal transducers. The modes of detection and sensitivity of the sensors are discussed. Finally, the challenges in the fabrication, obstacles in the clinical application, and future prospects are discussed.

Detection of Premalignant Cervical Lesions via Maackia amurensis Lectin-Based Biosensors

Early detection of premalignant cervical lesions is essential for improving cervical cancer outcomes; however, current screening methods frequently lack adequate sensitivity and specificity. This research introduces a diagnostic platform that integrates lectin-based biosensors with spectral and multivariate analysis. The biosensors are composed of gold nanoparticles (AuNPs) conjugated with Maackia amurensis (MAA) lectin, which selectively binds to α2,3-linked sialic acid. Validation was performed using cervical cancer cell lines (SiHa, HeLa, C33A), fibroblasts, and cervical scrapes, and specificity was verified by enzymatic removal of sialic acids. Spectral data were obtained using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and analyzed by principal component analysis (PCA). Application of PCA to the 1600–1350 cm−1 spectral region, using 99% confidence ellipses, enabled clear differentiation between samples negative and positive for intraepithelial lesions in a double-blind study of 58 patients. The MAA biosensors exhibited high sensitivity and specificity, comparable to established diagnostic methods. These results indicate that the combination of ATR-FTIR spectroscopy, MAA lectin-based biosensors, and chemometric analysis provides a robust and reliable approach for early detection of premalignant cervical lesions, with considerable potential to enhance patient outcomes.

Recent Advances in SAW Sensors for Detection of Cancer Biomarkers

Surface acoustic wave (SAW) sensor technology is a promising approach to diagnosing cancer through the detection of cancer biomarkers due to its high sensitivity, potential label-free operation, and fast response times, and, fundamentally, because it is a non-invasive technique in comparison with the current traditional diagnostic techniques for cancer. This review focuses on this application, and for this purpose, the recent literature on cancer biomarkers detected by this advanced technology has been compiled, including that on volatile organic compounds (VOCs) from exhaled breath and larger biomolecules such as proteins, DNA, and microRNAs in body fluids, which demonstrates its great versatility. The conventional techniques for cancer biomarker detection in biofluids, such as ELISA, PCR, SPR, and UV absorbance, exhibit limitations including high costs, slow response times, a reduced sensitivity, the need for specialized instrumentation, and the requirement for highly trained personnel. Different SAW sensor configurations are discussed with attention paid to their specific properties, wave propagation modes, and suitability for different environments. Detailed studies are reviewed, highlighting biomarkers for lung, colorectal, prostate, breast, and ovarian cancer diagnostics, as well as the detection of circulating tumor cells and cancerous cell growth. This review identifies current challenges, including optimizing sensitivity, addressing environmental interferences, and the need for clinical validation. Finally, future research directions are proposed, emphasizing the use of VOC biomarkers and the integration of SAW technology into hybrid systems and microfluidic platforms to enable the creation of scalable, non-invasive diagnostic tools for the detection of cancer in early stages, and, in this way, to minimize the morbidity and mortality associated with this disease.

Detection of Sialic Acid to Differentiate Cervical Cancer Cell Lines Using a Sambucus nigra Lectin Biosensor

Pap smear screening is a widespread technique used to detect premalignant lesions of cervical cancer (CC); however, it lacks sensitivity, leading to identifying biomarkers that improve early diagnosis sensitivity. A characteristic of cancer is the aberrant sialylation that involves the abnormal expression of α2,6 sialic acid, a specific carbohydrate linked to glycoproteins and glycolipids on the cell surface, which has been reported in premalignant CC lesions. This work aimed to develop a method to differentiate CC cell lines and primary fibroblasts using a novel lectin-based biosensor to detect α2,6 sialic acid based on attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and chemometric. The biosensor was developed by conjugating gold nanoparticles (AuNPs) with 5 µg of Sambucus nigra (SNA) lectin as the biorecognition element. Sialic acid detection was associated with the signal amplification in the 1500–1350 cm−1 region observed by the surface-enhanced infrared absorption spectroscopy (SEIRA) effect from ATR-FTIR results. This region was further analyzed for the clustering of samples by applying principal component analysis (PCA) and confidence ellipses at a 95% interval. This work demonstrates the feasibility of employing SNA biosensors to discriminate between tumoral and non-tumoral cells, that have the potential for the early detection of premalignant lesions of CC.

Application of Various Optical and Electrochemical Nanobiosensors for Detecting Cancer Antigen 125 (CA-125): A Review

Nowadays, diagnosing early-stage cancers can be vital for saving patients and dramatically decreases mortality rates. Therefore, specificity and sensitivity in the detection of cancer antigens should be elaborately ensured. Some early-stage cancers can be diagnosed via detecting the cancer antigen CA-125, such as ovarian cancer, and required treatments can be applied more efficiently. Thus, detection of CA-125 by employing various optical or electrochemical biosensors is a preliminary and crucial step to treating cancers. In this review, a diverse range of optical and electrochemical means of detecting CA-125 are reviewed. Furthermore, an applicable comparison of their performance and sensitivity is provided, several commercial detection kits are investigated, and their applications are compared and discussed to determine whether they are applicable and accurate enough.

An Immunosensor for the Determination of Cathepsin S in Blood Plasma by Array SPRi—A Comparison of Analytical Properties of Silver–Gold and Pure Gold Chips

The array SPR imaging (SPRi) technique is well suited to the determination of biomarkers in body fluids, called liquid biopsy. No signal enhancement or analyte preconcentration is required. With the aim of achieving signal enhancement and lowering the cost of a single determination, the replacement of gold-covered chips by silver–gold chips was investigated. The aim of this work was to investigate the analytical characteristics of a biosensor formed on a Ag/Au chip and to compare them with those of a biosensor formed on a gold chip. A biosensor for the determination of cathepsin S (Cath S) was chosen as an example. The biosensor consisted of the linker cysteamine and an immobilized rat monoclonal antibody specific for cathepsin S. Both biosensors exhibited a Langmuirian response to Cath S concentration, with linear response ranging from LOQ to 1.5 ng mL−1. The LOQ is 0.1 ng mL−1 for the biosensor formed on the Ag/Au chip, and 0.22 ng mL−1 for that formed on the gold chip. Recoveries and precision for medium and high Cath S concentrations were acceptable for both biosensors, i.e., precision better than 10% and recoveries within the range 102–105%. However, the results for the lowest Cath S concentration were better for the biosensor formed on the Ag/Au chip (9.4 and 106% for precision and recovery, respectively). Generally, no significant differences in analytical characteristics were observed between the Ag/Au and Au chips. The two biosensors were also compared in the determination of Cath S in real samples. Nine plasma samples from healthy donors and nine from patients with ovarian cancer were analyzed for Cath S concentration with the biosensors formed on Ag/Au and Au chips. The results obtained with the two biosensors were very similar and show no significant differences on the Bland–Altman plot. The Cath S concentration in the blood plasma of ovarian cancer patients was elevated by one order of magnitude as compared with the control (12.6 ± 3.6 vs. 1.6 ± 1.2 ng mL−1).

Aptamer-Based Graphene Field-Effect Transistor Biosensor for Cytokine Detection in Undiluted Physiological Media for Cervical Carcinoma Diagnosis

Personalized monitoring of disease biomarkers is of great interest in women’s health. However, existing approaches typically involve invasive inspection or bulky equipment, making them challenging to implement at home. Hence, we present a general strategy for label-free and specific detection of disease biomarkers in physiological media using an aptamer-based biosensor. The biosensor is a graphene field-effect transistor that involves immobilizing the aptamer and a biomolecule-permeable polyethylene glycol (PEG) layer on the graphene surface. The aptamer is capable of specifically binding with the target biomarker, thus inducing a change in the sensing responses. The PEG layer can effectively reduce the nonspecific adsorption of nontarget molecules in the solution, and increase the effective Debye screening length in the region directly adjacent to the graphene. In this work, studies of a biosensor with modification of the aptamer and PEG show that cervical carcinoma biomarkers such as tumor necrosis factor-α and interleukin 6 can be sensitively and specifically detected in undiluted physiological media, with detection limits as low as 0.13 pM for TNF-a and 0.20 pM for IL-6. This work presents a significant method for the general application of the biosensor for disease diagnosis in women’s health.

Comparison of Survivin Determination by Surface-Enhanced Fluorescence and Raman Spectroscopy on Nanostructured Silver Substrates

Survivin belongs to a family of proteins that promote cellular proliferation and inhibit cellular apoptosis. Its overexpression in various cancer types has led to its recognition as an important marker for cancer diagnosis and treatment. In this work, we compare two approaches for the immunochemical detection of survivin through surface-enhanced fluorescence or Raman spectroscopy using surfaces with nanowires decorated with silver nanoparticles in the form of dendrites or aggregates as immunoassays substrates. In both substrates, a two-step non-competitive immunoassay was developed using a pair of specific monoclonal antibodies, one for detection and the other for capture. The detection antibody was biotinylated and combined with streptavidin labeled with rhodamine for the detection of surface-enhanced fluorescence, while, for the detection via Raman spectroscopy, streptavidin labeled with peroxidase was used and the signal was obtained after the application of 3,3′,5,5′-tetramethylbenzidine (TMB) precipitating substrate. It was found that the substrate with the silver dendrites provided higher fluorescence signal intensity compared to the substrate with the silver aggregates, while the opposite was observed for the Raman signal. Thus, the best substrate was used for each detection method. A detection limit of 12.5 pg/mL was achieved with both detection approaches along with a linear dynamic range up to 500 pg/mL, enabling survivin determination in human serum samples from both healthy and ovarian cancer patients for cancer diagnosis and monitoring purposes.

Two Biosensors for the Determination of Interleukin-6 in Blood Plasma by Array SPRi

Interleukin-6 (IL-6) is a biomarker of inflammation, the advanced stage of COVID-19, and several cancers, including ovarian cancer. Two biosensors for the determination of IL-6 in blood plasma by array SPRi have been developed. One of these biosensors consists of the mouse monoclonal anti-IL-6 antibody as the receptor immobilized via the cysteamine linker. The second contains galiellalactone as the receptor, being an inhibitor specific for IL-6, immobilized via octadecanethiol (ODM) as the linker. Both biosensors are specific for IL-6. The biosensor with the antibody as the receptor gives a linear analytical response between 3 (LOQ) and 20 pg mL−1 and has a precision between 8% and 9.8% and recovery between 97% and 107%, depending on the IL-6 concentration. The biosensor with galiellalactone as the receptor gives a linear analytical response between 1.1 (LOQ) and 20 pg mL−1, and has a precision between 3.5% and 9.3% and recovery between 101% and 105%, depending on IL-6 concentration. Both biosensors were validated. Changes in IL-6 concentration in blood plasma before and after resection of ovarian tumor and endometrial cyst, as determined by the two developed biosensors, are given as an example of a real clinical application.

Detection of the Ovarian Cancer Biomarker Lysophosphatidic Acid in Serum

Lysophosphatidic acid (LPA) is present during the medical condition of ovarian cancer at all stages of the disease, and, therefore possesses considerable potential as a biomarker for screening its presence in female patients. Unfortunately, there is currently no clinically employable assay for this biomarker. In the present work, we introduce a test based on the duel protein system of actin and gelsolin that could allow the quantitative measurement of LPA in serum samples in a biosensing format. In order to evaluate this possibility, actin protein was dye-modified and complexed with gelsolin protein, followed by surface deposition onto silica nanoparticles. This solid-phase system was exposed to serum samples containing various concentrations of LPA and analyzed by fluorescence microscopy. Measurements conducted for the LPA-containing serum samples were higher after exposure to the developed test than samples without LPA. Early results suggest a limit of detection of 5 μM LPA in serum. The eventual goal is to employ the chemistry described here in a biosensor configuration for the large population-scale, rapid screening of women for the potential occurrence of ovarian cancer.

An Interfacial Affinity Interaction-Based Method for Detecting HOTAIR lncRNA in Cancer Plasma Samples

Long non-coding RNA Homeobox transcript antisense intergenic RNA (HOTAIR) is recognized as a participant in different processes of normal cell development. Aberrant overexpression of HOTAIR contributes to the initiation, growth, and invasiveness of ovarian cancer. Using the affinity interaction of target HOTAIR lncRNA sequences towards a screen-printed gold electrode (SPE-Au), herein we report on a novel, rapid and simple method to detect HOTAIR sequences. HOTAIR lncRNA sequences were first extracted from ovarian cancer cell lines and patient plasma samples and were magnetically captured and purified by complimentary capture probe-functionalized magnetic beads. Isolated target HOTAIR lncRNAs were directly adsorbed onto unmodified screen-printed gold electrodes (SPE-Au) for direct quantification with [Fe(CN)6]3−/4− redox couple. Our assay achieved a linear dynamic range of 100 nM and 1 pM for detecting pre-clinical model HOTAIR lncRNA samples (%RSD ≤ 5%, for n = 3) and was highly specific, showing clear distinction between HOTAIR lncRNA targets and non-specific miR-891 and miR-486 (100 nM) (%RSD ≤ 5%, for n = 3). The method was tested using ovarian cancer-specific cell lines (SKOV3 and OVCAR3) and mesothelial cell line (MeT-5A)-derived lncRNAs. The analytical performance of our method was validated using RT-qPCR. Finally, the method was tested using clinical samples from ovarian cancer patients and the resulting electrochemical responses show a clear distinction between the ovarian carcinoma and benign samples.

Folic Acid-Modified Fluorescent-Magnetic Nanoparticles for Efficient Isolation and Identification of Circulating Tumor Cells in Ovarian Cancer

Ovarian cancer (OC) is a lethal disease occurring in women worldwide. Due to the lack of obvious clinical symptoms and sensitivity biomarkers, OC patients are often diagnosed in advanced stages and suffer a poor prognosis. Circulating tumor cells (CTCs), released from tumor sites into the peripheral blood, have been recognized as promising biomarkers in cancer prognosis, treatment monitoring, and metastasis diagnosis. However, the number of CTCs in peripheral blood is low, and it is a technical challenge to isolate, enrich, and identify CTCs from the blood samples of patients. This work develops a simple, effective, and inexpensive strategy to capture and identify CTCs from OC blood samples using the folic acid (FA) and antifouling-hydrogel-modified fluorescent-magnetic nanoparticles. The hydrogel showed a good antifouling property against peripheral blood mononuclear cells (PBMCs). The FA was coupled to the hydrogel surface as the targeting molecule for the CTC isolation, held a good capture efficiency for SK-OV-3 cells (95.58%), and successfully isolated 2–12 CTCs from 10 OC patients’ blood samples. The FA-modified fluorescent-magnetic nanoparticles were successfully used for the capture and direct identification of CTCs from the blood samples of OC patients.

Cervical Imaging in the Low Resource Setting: A Review

Cervical cancer is one of the most significant global health inequities of our time and is the fourth most common cancer in women worldwide, disproportionally affecting developing countries where the disease burden is 84%. Sometimes referred to as preventable cancer, it progresses slowly, providing a window of time for routine screening in which pre-cancerous lesions can be identified and treated. The high mortality rate can be attributed to many reasons, including the high cost of cytology-based screening, lack of human resources to conduct screening, and inadequate preventive medicine services and systems. Due to its slow progression, early intervention is feasible with appropriate screening. However, the standard screening procedures require access to lab-based tests and physician expertise. Several imaging devices have been introduced in the literature to aid cervical screening in low-resource settings. This review details the instrumentation and clinical testing of devices currently deployed in low-resource locations worldwide. The devices’ imaging, portability, illumination, and power requirements (among other metrics) are documented with specifics of human pilot studies conducted with these tools.

APTES-Modified Remote Self-Assembled DNA-Based Electrochemical Biosensor for Human Papillomavirus DNA Detection

High-risk human papillomavirus (HPV) infection is an important cause of cervical cancer formation; therefore, being able to detect high-risk HPV (e.g., HPV-16) is important for the early treatment and prevention of cervical cancer. In this study, a combination of a 3-aminopropyltriethoxysilane (APTES) modified gold electrode and a super sandwich structure was creatively developed, resulting in the development of a biosensor that is both sensitive and stable for the detection of HPV-16. The electrochemical biosensor possesses a lower detection limit compared with previous studies with an LOD of 5.475 × 10−16 mol/L and it possesses a wide linear range from 1.0 × 10−13 mol/L to 1.0 × 10−6 mol/L (R2 = 0.9923) for the target DNA. The experimental data show that the sensor has good stability, and there is no significant decrease in the current response value after 7 days in the low-temperature environment. In addition, the sensor proved to be a powerful clinical tool for disease diagnosis because it showed good interference resistance in complex human serum samples.

Chemiluminescent Biosensor Utilizing Magnetic Particles for the Detection of Ovarian Cancer Biomarker Lysophosphatidic Acid

Lysophosphatidic acid (LPA) is a cell-signaling lipid that has been proposed as an early-stage biomarker for ovarian cancer (OC). Diagnosing OC in Stage I is critical to improving patient outcomes, increasing the survival rate from 30% (when diagnosed in late stages of the disease) to over 90%. This significant improvement is due to the success of early interventions; however, current diagnostic methods are not as effective at early-stage detection, with only 15% of cases diagnosed in Stage I and over 70% diagnosed in Stage III or IV. There is a strong need for LPA detection that is sensitive, specific, rapid, low-cost, and automated to truly validate its effectiveness as a diagnostic characteristic for OC. We report the preliminary development and characterization of one such biosensor, which makes use of the advantages of magnetic particles and chemiluminescence for quick, sensitive detection of LPA. The sensor has proven to be viable, with a positive response to LPA concentration, a measurement time of 5 s after incubation, and an LOD of 3.5 nM.